Eukaryotic genes, including most human genes, are interrupted by numerous introns. After transcription of such genes, the introns are excised in two phosphoryl transfer reactions catalyzed by the spliceosome, a macromolecular machine composed of both protein and RNA. In the first reaction, the 2'hydroxyl of an intronic adenosine attacks the 5'splice site cleaving the intron from the 5'exon. In the second reaction, the newly- formed 3'hydroxyl of the liberated 5'exon attacks the 3'splice site, excising the intron and ligating the flanking exons. The RNA components of the spliceosome have been implicated in both recognizing introns and catalyzing intron excision. Our long-term objective is to determine the mechanism by which the spliceosome catalyzes pre-mRNA splicing and in particular to define the role of RNA in catalysis, both in structural and functional terms. While reductionist approaches have revealed catalytic activities of the spliceosomal RNAs, these reactions are inefficient and incompletely characterized. Consequently, a mechanistic understanding of pre-mRNA splicing requires an investigation of the spliceosome itself. Interestingly, group II introns splice by a pathway indistinguishable from the spliceosome, and both enzymes share common RNA features. A recent crystal structure of a group II intron reveals two bound metals, suggesting metal ligands in the spliceosome and a mechanism for catalysis by both group II introns and the spliceosome. Indeed, using state-of-the-art chemical approaches, our previous studies have implicated metal-based catalysis in both steps of splicing and our work and that of others has implicated spliceosomal RNAs as catalytic metal ligands. Further, our recent discovery of fidelity mechanisms in vitro that impose high stringency on the chemistry of splicing now provides a strategy to relax these constraints and to more broadly investigate catalysis. Our near-term goal is to investigate the roles of metals in catalyzing pre-mRNA splicing, the identity of the ligands for such metals and the RNA structure required for metal binding and catalysis. Specifically, we aim (i) to investigate the role of metals and metal ligands in exon ligation, (ii) to investigate the role of metals and metal ligands in 5'splice site cleavage and (iii) to investigate the role of RNA tertiary interactions in promoting catalysis. We propose to accomplish these aims through a unique collaboration that allows a combined approach of chemistry, biochemistry and molecular genetics. We will utilize the model organism budding yeast, which allows for both biochemical and genetic studies of pre-mRNA splicing. Considering the potential similarity between the catalytic mechanisms of the spliceosome and group II introns, this work will have important implications for understanding the evolutionary origins of the spliceosome. Given that at least 15% of human diseases result from errors in splicing, this work will also illuminate the inner workings of a machine that is essential to the well- being of humans. PUBLIC HEALTH RELEVANCE: It has been estimated that at least 15% of human diseases result from defects in splicing. Our ability to treat such diseases will depend, in large part, on our understanding of the complexities of the splicing machinery. In its attempt to identify the essential catalytic elements of the spliceosome, this project will provide novel insights into the fundamental mechanisms that must function properly in healthy human cells and thereby illuminate possible mechanisms for disease as well as potential strategies for treatment.